Metabolomics is a powerful new systems biology tool that is capable of simultaneously investigating multiple biological pathways, detecting and diagnosing a disease and evaluating the efficacy of a therapy at an early stage. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the leading metabolomics tools. Tissue metabolic profiling is of significance because a disease is often associated with a localized tissue/organ malfunction. The technique of high resolution magic angle spinning (hr-MAS) has been increasingly used for metabolic profiling of intact tissues with notable successes. However, the requirement of tissue mass of 10- 30mg or more for standard hr-MAS NMR metabolomics analysis may present a problem in studying small laboratory animals such as mice, where animals need to be sacrificed to obtain adequate amount of tissue for subsequent analysis. Furthermore, a tissue mass of 10-30mg could encompass many different cell types and study on a smaller sample size would be desired in terms of cell biochemistry. The goal of our research is to develop a novel high resolution, high sensitivity method for NMR metabolomics investigations of tissue samples with mass/volume as small as 300<g/300nL. The outcome of the project will make it possible to carry out a continued investigation on a single small laboratory animal over time using minimally invasive tissue biopsy samples. To reach our goal, we have formulated two specific Aims. Aim 1: Development of a slow magic angle sample spinning NMR probe with sample volume as small as 300 nanoliters (nL). We will use a combination of micro-RF-coil with magnetic susceptibility matched wires, and capillary sample tube to obtain the optimal sensitivity. The spectral resolution will be further enhanced by integrating our established slow-MAS 1H NMR method applied at a sample spinning rate of about 80Hz. With slow-MAS technique, the line broadening due to magnetic susceptibility gradients within tissue cells, between the sample and the sample tube are eliminated, giving rise to a high resolution 1H NMR spectrum. We will expand the applicability of the probe such that tissue samples from 300nL to several mL can also be investigated. Initially, a probe will be built to operate at 7.05T field, followed by a second probe that will operate at 11.7 T field so the potential advantages of slow-MAS at higher magnetic field strengths can be explored. Aim 2: Application of the nL slow-MAS probe. We will carry out a comprehensive NMR metabolomics investigation using minimally invasive biopsy muscle samples of nL in volume (including the use of blood and non-invasive urine samples of <l or less as a supplementary method to standard NMR metabolomics) obtained from normal vs obese C57BL/6 mice from about 6 weeks to about 16 weeks of age to identify possible metabolite biomarkers that are related to obesity. The time variation of metabolite biomarkers for each animal will be determined. The results will be compared between the animals within the same group, from which the normal biological variations will be determined and the advantages of following a single animal over time period will be demonstrated. PUBLIC HEALTH RELEVANCE: We propose to develop a novel slow magic angle sample spinning probe that is capable of high resolution and high sensitivity metabolic profiling on biological samples, in particular tissue samples, with volume as small as 300 nanoliters (nL) to sample volume as large as a few milliliters. The nL capability will make it possible to follow the metabolic changes through a continued investigation on a single small laboratory animal, and ultimately on a patient, over a long period of time using minimally invasive tissue biopsy and blood samples.